Composite modified beta-lactoglobulin as well as preparation method and application thereof

Abstract

The invention discloses composite modified beta-lactoglobulin as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving beta-lactoglobulin powder in deionized water to form a beta-lactoglobulin solution; the preparation method comprises the following steps: sequentially carrying out microjet treatment and succinylation treatment to obtain microjet-assisted acylated beta-lactoglobulin, then dialyzing the acylated beta-lactoglobulin solution for purification treatment, and finally freeze-drying to obtain the composite modified acylated beta-lactoglobulin powder. The beta-lactoglobulin is subjected to composite modification by fusing microjet and succinylation, the compact structure of the protein is expanded by the microjet, reaction active sites in the protein are exposed, the solubility of the protein is improved, and meanwhile, convenience is provided for subsequent chemical modification by breakage of non-covalent bonds. The introduction of the succinyl group adjusts the surface property of the protein and improves the emulsifying property of the beta-lactoglobulin. And meanwhile, the oxidation resistance of the beta-lactoglobulin is enhanced due to the oxidation resistance of the succinyl.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a composite modified beta-lactoglobulin and a preparation method and application thereof. Background Art

[0002] β-lactoglobulin (β-LG) is mainly found in milk and other mammalian milk. It is the main component of whey protein, accounting for about 50% of the total whey protein. It exists in the form of dimers composed of two identical polypeptide chains, each with about 160-162 amino acid residues and an overall molecular weight of about 18-25 kDa. The typical α / β curled structure formed by two disulfide bonds is essential for maintaining the stability and function of the protein. At the same time, β-lactoglobulin is rich in a variety of essential amino acids, can promote protein synthesis in the human body, enhance the body's immunity, and has antibacterial and antiviral, antioxidant, blood sugar regulation, growth and development, and sleep improvement functions. In addition, β-lactoglobulin hydrolyzate also has the effects of lowering cholesterol, improving intestinal flora, and promoting mineral absorption, and is easy for the human body to digest and absorb. Therefore, a reasonable intake of β-lactoglobulin can give full play to its rich nutritional value. However, for certain populations, especially infants and those with sensitive immune systems, β-lactoglobulin may cause allergic reactions. Despite potential limitations, β-lactoglobulin still shows broad application potential and value in food processing, nutritional supplementation and medicine due to its unique structure and functional properties.

[0003] Succinic anhydride, also known as succinic anhydride (SA), is an organic compound (C4H4O3) with a unique cyclic anhydride. With its unique chemical properties, it occupies an important position in the pharmaceutical, chemical and food industries. In medicine, it is a key raw material for many drugs; in the chemical field, it plays an important role as a chemical intermediate and resin hardener; in the food industry, it is used as an acidulant, thickener and flavoring agent. In addition, succinic anhydride is often used to modify proteins to improve the functional activity of proteins, including solubility, thermal stability and emulsification. Studies have shown that succinic anhydride mainly covalently binds to protein amino groups (especially the ε-amino group of lysine) to form amide bonds, significantly changing the spatial structure of the protein surface, stretching the α-helix and β-folded structures, destroying hydrogen bonds and hydrophobic forces, and adjusting the protein structure, thereby affecting its functional activity. However, since β-lactoglobulin is a globular protein, the internal lysine is difficult to directly access, and its structure needs to be partially unfolded to allow succinic anhydride to enter the protein or expose the internal active sites, thereby maximizing the reaction modification effect and better optimizing its functional properties. Therefore, composite processing technology is particularly important in protein modification, laying a solid foundation for achieving a leap in protein performance.

[0004] Protein modification refers to the regulation of protein structure by physical, chemical or biological methods, thereby giving it better functional properties. Among them, chemical modification such as phosphorylation, glycosylation, and polyphenolization regulates the surface charge and hydrophilic-hydrophobic properties of proteins by introducing chemical bonds; biological modification mainly uses the efficient catalysis of enzymes to achieve specific hydrolysis or cross-linking of proteins, optimizing their solubility and emulsification; physical modification can directly act on proteins by physical means without introducing impurities, changing their surface charge and structure. However, a single modification treatment is often difficult to deeply optimize protein performance. In this context, composite modification technology came into being. It cleverly integrates physical, chemical or biological modification methods, combines two or more methods, and achieves multi-dimensional and deep regulation of proteins through synergistic effects. For example, high pressure can generate strong pressure through high pressure, break the tight binding between protein molecules, and promote the partial unfolding of their structure. This process is efficient and effective, providing favorable conditions for subsequent reactions, enabling it to significantly improve the antioxidant and emulsifying properties of proteins. Summary of the invention

[0005] The present invention aims to overcome the disadvantages of β-lactoglobulin, such as low solubility, emulsification and antioxidant properties, which limit its application in food and biological fields, such as the buried functional groups due to its compact structure. Therefore, a composite modified β-lactoglobulin and its preparation method and application are provided, and the composite modified β-lactoglobulin is applied to dairy products, sports nutrition products, functional foods, baked foods and other foods.

[0006] To achieve the above-mentioned object of the invention, the present invention is implemented by the following technical scheme: a method for preparing a composite modified β-lactoglobulin, comprising the following steps:

[0007] (1) dissolving β-lactoglobulin powder in deionized water to form a β-lactoglobulin solution, and then subjecting the solution to microfluidization treatment to obtain a microfluidization-modified β-lactoglobulin solution;

[0008] (2) The microfluidization-modified β-lactoglobulin solution is subjected to succinylation treatment to obtain microfluidization-assisted acylated β-lactoglobulin, and the acylated β-lactoglobulin solution is then dialyzed for purification, and finally freeze-dried to obtain composite modified acylated β-lactoglobulin.

[0009] Furthermore, the preparation method of the β-lactoglobulin powder comprises:

[0010] β-lactoglobulin is extracted from whey protein, dialyzed at room temperature, and then freeze-dried to obtain highly purified β-lactoglobulin powder.

[0011] Further, β-lactoglobulin is extracted from whey protein, dialyzed at room temperature, and then freeze-dried to obtain highly purified β-lactoglobulin powder, including:

[0012] The whey protein solution with a concentration of 4.0-6.0wt% was adjusted to pH 3.5-4 with 1M citric acid, heated in a water bath at 30-40°C for 60-80min, and then centrifuged to obtain the supernatant; 7.0-7.5wt% sodium chloride was added for salting out for 50-70min, and then the supernatant was collected by centrifugation, and the process was repeated once; the solution was dialyzed for 48h and then freeze-dried for use.

[0013] Among them, whey protein concentrations of 4.0~6.0wt% have good extraction efficiency, while costs are controlled and purity is guaranteed. Concentrations below 4.0wt% will reduce extraction efficiency and increase costs; concentrations above 6wt% will increase processing difficulty, resulting in decreased purity, and increased equipment load will lead to increased costs. The isoelectric point of β-lactoglobulin is usually between 5.1 and 5.3. Adjusting the pH to 3.5~4 means that β-lactoglobulin is in an acidic environment below the isoelectric point. In this environment, β-lactoglobulin has low solubility, which promotes its separation and purification. At the same time, in a low pH environment, some impurities with similar properties to β-lactoglobulin will also precipitate, thereby improving the extraction purity. Citric acid, as a natural additive, is safe and environmentally friendly, in line with sustainable development, and its weak acidity can keep the nutrients intact and optimize the subsequent salting-out effect.

[0014] A temperature of 30~40℃ helps to promote the separation of β-lactoglobulin from other protein components, reduce interference from impurities, and most importantly, maintain the functional activity of the protein. Temperatures above 40℃ may cause protein denaturation, reduce its biological activity and function, increase impurities, and increase energy consumption. Temperatures below 30℃ may reduce extraction efficiency and increase the risk of microbial contamination. Heating for 60~80min can promote the movement of protein molecules while maintaining protein activity and stable structure. Temperatures below 60min may result in incomplete extraction and residual impurities; temperatures above 80min may result in local overheating, leading to protein denaturation, increased energy consumption, and increased equipment burden.

[0015] When the salting-out concentration is 7-7.5wt%, it can maintain both the low solubility and biological activity of β-lactoglobulin. A concentration higher than 7.5wt% will cause excessive aggregation of the protein to form a precipitate, affecting its biological activity and function. A concentration lower than 7wt% will result in an insignificant decrease in protein solubility, making it difficult to achieve the ideal separation effect. The best salting-out effect is achieved by performing the salting-out twice within 50-70min, and β-lactoglobulin is fully precipitated. When the treatment time exceeds 140min at a time, the protein is exposed to a high salt concentration environment for a long time, increasing the risk of denaturation. At the same time, it may re-dissolve or undergo other adverse reactions, resulting in reduced separation and purification efficiency and waste of resources. When the treatment time is less than 60min, the salt ions do not fully destroy the hydration layer of the protein, resulting in incomplete salting-out, poor separation effect, and the addition of additional steps, which increases costs. Therefore, performing the salting-out twice can reduce the impact on the protein structure and fully salt out.

[0016] Furthermore, the pH of the whey protein solution is adjusted to 3.5-4 with 1M citric acid.

[0017] Furthermore, the concentration of the β-lactoglobulin solution in step (1) is 0.5-1.5 wt %.

[0018] Among them, the most suitable concentration of β-lactoglobulin is 0.5~1.5wt%. A concentration higher than 1.5wt% will increase the difficulty of processing, the protein solution will become viscous, and the equipment will be worn or blocked when passing through the high-pressure microjet. At the same time, too high a concentration will cause some β-lactoglobulin to aggregate excessively, affecting the emulsifying activity of the protein. A concentration lower than 0.5wt% will result in insignificant modification treatment effects and complicated operations, and will have little effect on the emulsifying properties and antioxidant properties of the protein. Because low concentrations require more precise control conditions and more complex operating procedures, the economic benefits are low.

[0019] Furthermore, the pressure of the microfluidization treatment in step (1) is 50-100 MPa.

[0020] Among them, high-pressure microfluidics can disperse the particles of β-lactoglobulin, reduce the particle size, promote its solubility and dispersibility, and at the same time, the structure is obvious, exposing more active sites, promoting subsequent acylation reactions, and improving protein emulsification and antioxidant properties. When the pressure is greater than 100MPa, the protein structure is excessively destroyed, resulting in agglomeration. At the same time, some active sites are destroyed, affecting the acylation reaction and reducing antioxidant activity. When the pressure is less than 50MPa, the lower pressure is not enough to break the intermolecular forces, and its structure cannot be unfolded, solubility is reduced, emulsification activity is affected, and it is difficult to achieve the ideal effect.

[0021] Furthermore, the step (2) comprises:

[0022] The β-lactoglobulin solution modified by microfluidization in step (1) is added with succinic anhydride at a concentration of 4% to 20% (w / w), and the solution is subjected to constant temperature magnetic stirring in a water bath. The pH of the acylation reaction is always maintained at 8 to 8.5, the temperature is 35 to 50° C., and the solution is stirred for 120 to 150 minutes to form a microfluidization-assisted acylated β-lactoglobulin solution. The solution is then dialyzed for 48 hours to remove excess succinic anhydride and impurities to obtain a purified microfluidization-assisted acylated β-lactoglobulin solution. The purified microfluidization-assisted acylated β-lactoglobulin solution is subjected to vacuum freeze-drying to obtain a powdered sample, i.e., a composite modified β-lactoglobulin.

[0023] Among them, the concentration of succinic anhydride in 4%~20% includes three levels of acylation degree: low, medium and high. When the mass ratio is lower than 4%, the protein acylation degree is not obvious, the reaction is incomplete, and the antioxidant capacity and emulsification properties of the protein cannot be significantly improved. When the mass ratio is higher than 20%, the protein acylation degree does not change significantly, and saturation begins to appear, resulting in waste of resources and increased costs. During the acylation process, the temperature is controlled at 37~50℃, which is conducive to the control of the reaction and the formation of products, and will not have a significant effect on the stability of most proteins. When the acylation temperature is higher than 50℃, the protein is prone to denaturation and aggregation, the solubility is reduced, and the by-products may increase, resulting in reduced sample purity. When the acylation temperature is lower than 37℃, the reaction rate is slow, the efficiency is low, the time is prolonged, the energy consumption is large, and the cost is increased. When the pH of the acylation reaction is between 8~8.5, it will promote the deprotonation of the protein amino group and increase its reaction activity. At the same time, this pH range will not cause significant damage to the protein structure. A pH higher than 8.5 will lead to excessive deprotonation of the amino group, increase the nonspecific reaction, reduce the purity of acylated β-lactoglobulin and thus reduce its emulsifying activity. A pH lower than 8 will reduce the reaction activity, increase the degree of amino protonation, reduce the reaction rate and thus affect the emulsifying activity and antioxidant properties of β-lactoglobulin.

[0024] Further, the pH is adjusted to 8-8.5 by using a sodium hydroxide solution having a concentration of 1-2 M.

[0025] Among them, the appropriate concentration can more efficiently and quickly adjust the pH value, reduce unnecessary side reactions, and thus improve the purity and quality of the product. A concentration greater than 2M will increase the risk of side reactions and affect the reaction rate. At the same time, the high alkaline environment will change the protein structure, affecting its solubility and emulsification activity. In addition, there will be certain safety hazards. A concentration lower than 1M will result in a long adjustment time, reduced reaction efficiency, reduced accuracy, and the inability to reach the optimal pH of the reaction in time, which will reduce the reaction efficiency and further affect its antioxidant capacity.

[0026] Furthermore, the composite modified β-lactoglobulin is prepared according to the above preparation method.

[0027] Furthermore, the composite modified β-lactoglobulin prepared according to the above preparation method or the application of the above composite modified β-lactoglobulin in protein processing function.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention uses a high-pressure microfluidization-assisted succinylation reaction to perform composite modification on β-lactoglobulin. Since the succinyl group is negatively charged in an alkaline environment, it effectively increases the absolute potential of β-lactoglobulin, promotes the repulsion between proteins, and enhances its solubility. At the same time, succinic anhydride has good lipophilic properties and reacts with proteins to form an acylated β-lactoglobulin complex, which significantly improves its emulsification properties.

[0030] (2) The present invention significantly improves the binding of β-lactoglobulin and succinic anhydride with the assistance of high-pressure microfluidization, and significantly enhances the antioxidant properties of β-lactoglobulin through chemical modification. The preparation process of the complex is simple, efficient and low-cost, so it shows good application potential and development prospects in the field of food processing.

[0031] (3) Acylation changes the charge of β-lactoglobulin, thereby weakening the interaction between molecules and making it more conducive to the formation of a stable interfacial film. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0033] Figure 1 is the acylation reaction equation of the composite modified β-lactoglobulin;

[0034] Figure 2 The present invention is a preparation process of composite modified β-lactoglobulin;

[0035] Figure 3 is the solubility of the composite modified β-lactoglobulin;

[0036] Figure 4 The emulsifying properties of the composite modified β-lactoglobulin;

[0037] Figure 5 The antioxidant properties of the composite modified β-lactoglobulin;

[0038] Figure 6 The foaming properties of composite modified β-lactoglobulin. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0040] Protein is an indispensable component of the human body. It participates in various metabolic processes in the body, and its complex and variable structure confers significant functional characteristics. β-Lactoglobulin is one of the main proteins in mammalian milk. It has multiple physiological activities such as lowering cholesterol, anti-oxidation, and antiviral. Its hydrolyzate has the ability to lower blood pressure, enhance the body's immunity, and promote mineral absorption. β-Lactoglobulin is often used as a nutritional additive, functional food ingredient, emulsifier, and new food ingredient after processing and modification in the food industry. However, natural β-lactoglobulin has a spherical structure, which limits its application to a certain extent. At the same time, its structure is easily affected by environmental factors such as temperature and pH value, resulting in unstable function. In order to overcome these limitations, it needs to be appropriately modified and its structure adjusted by scientific methods to improve its stability and functionality, thereby expanding its application potential in food and other fields.

[0041] Traditional protein modification includes physical, chemical and biological modification. Physical modification includes heat treatment, freezing, radiation, etc., which can change the structure of β-lactoglobulin and improve its solubility, but the optimization effect on its functional properties is not significant and it is difficult to achieve multiple optimization goals. Chemical modification includes glycosylation, polyphenolization, etc., which can change the functional properties of β-lactoglobulin by introducing new chemical groups, but there is a limitation of difficulty in controlling the degree of modification. Biological modification includes enzyme catalysis, microbial modification, etc., which introduces or changes the chemical groups of β-lactoglobulin through biocatalysts to adjust its functional properties, but may face limitations such as slow reaction rate, poor enzyme stability and high cost. In order to overcome the shortcomings of these single modifications, composite modification came into being. By combining different modification technologies, the structure and function of β-lactoglobulin can be comprehensively regulated to achieve a more optimized modification effect.

[0042] High-pressure microfluidization is an emerging protein modification technology. It uses high-speed flow and shear force to destroy the original structure of protein solution by spraying it through a tiny nozzle under high pressure. This method may deconstruct and reshape the secondary structure of β-lactoglobulin (such as α-helix and β-fold), thereby changing its overall conformation and generating more active binding sites or structural units, thereby promoting the subsequent acylation reaction. In addition, high-pressure microfluidization also causes the non-covalent bonds (such as hydrogen bonds and ionic bonds) of β-lactoglobulin to break and reorganize, and causes the molecules to depolymerize or aggregate to form a new spatial conformation. These structural changes will cause significant changes in its physical and chemical properties, such as improving its solubility and emulsification. Compared with traditional physical modification methods, high-pressure microfluidization not only has a shorter processing time, but also has more significant effects, and can significantly improve the functional properties of β-lactoglobulin in a short time. These changes and the resulting changes in physical and chemical properties provide new possibilities and optimization space for the application of β-lactoglobulin in food, medicine, biomaterials and other fields.

[0043] Succinic anhydride modified β-lactoglobulin introduces acyl groups through acylation reaction, resulting in the replacement of ε-amino groups by acyl groups, which significantly changes the chemical properties and spatial conformation of the protein. Acyl groups bind to protein amino groups through covalent bonds, while breaking and reorganizing non-covalent interactions, causing its structure to be destroyed or reshaped. This reaction can significantly enhance its stability and change its chemical properties, such as hydrophilicity and hydrophobicity, thereby improving its emulsifying properties. At the same time, the introduced acyl groups may increase the reactivity of the protein and promote the exposure of its active sites, thereby improving its antioxidant capacity and achieving the effect of optimizing function.

[0044] In summary, the combination of physical modification and chemical modification shows significant advantages in protein modification, which can achieve precise regulation of the structure of β-lactoglobulin and effectively optimize its functional properties. Physical modification methods, such as heat treatment and high pressure treatment, can change the spatial structure of β-lactoglobulin, thereby affecting its solubility and stability. Heat treatment can cause partial denaturation of β-lactoglobulin, and high pressure treatment may cause the structure of β-lactoglobulin to tighten or refold. Combined with chemical modification techniques, such as glycosylation, polyphenolization and acylation, the functional properties of β-lactoglobulin can be further enhanced. Glycosylation can improve its stability and solubility, polyphenolization can provide additional antioxidant properties, and acylation can change the hydrophilic-hydrophobic and antioxidant properties of β-lactoglobulin.

[0045] like Figure 1As shown, the present invention innovatively combines high-pressure microfluidization and succinylation to carry out composite modification of β-lactoglobulin. High-pressure microfluidization unfolds the tight structure of the protein, exposing the reactive sites inside the protein and improving the solubility of the protein. At the same time, the breaking of non-covalent bonds (such as hydrogen bonds and van der Waals forces) facilitates subsequent chemical modification. Furthermore, the introduction of hydrophilic succinyl groups adjusts the surface properties of the protein and significantly improves the emulsifying properties of β-lactoglobulin. At the same time, the antioxidant properties of succinyl groups also give β-lactoglobulin enhanced antioxidant properties.

[0046] Example 1

[0047] A method for preparing a composite modified β-lactoglobulin comprises the following steps:

[0048] (S. 1) Adjust the pH of whey protein with a concentration of 4.0-6.0wt% to 3.5-4 with 1M citric acid, heat in a water bath at 30-40℃ for 60-80min, centrifuge to obtain the supernatant, add 7.0-7.5wt% sodium chloride for salting out for 50-70min, centrifuge to collect the supernatant, repeat once, dialyze the solution for 48h, and freeze-dry for later use;

[0049] (S. 2) dissolving the β-lactoglobulin powder separated in step (S. 1) in deionized water to obtain a 1 wt% β-lactoglobulin solution, subjecting the solution to high-pressure microfluidization treatment at a pressure of 50 MPa, and performing a cycle operation for 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin;

[0050] (S. 3) Add 4% (w / w) succinic anhydride to the β-lactoglobulin solution modified by high-pressure microfluidization in step (S. 2), and stir in a water bath under constant temperature magnetic stirring. The pH of the acylation reaction is always maintained at 8-8.5, and the stirring is performed for 120-150 minutes to form succinylated β-lactoglobulin. The excess succinic anhydride and impurities are removed by dialysis for 48 hours to obtain a purified succinylated β-lactoglobulin solution. The solution is subjected to vacuum freeze-drying to obtain a powdered sample (i.e., composite modified acylated β-lactoglobulin powder), and after grinding, composite modified β-lactoglobulin particles with uniform particles are obtained.

[0051] Example 2

[0052] A method for preparing a composite modified β-lactoglobulin comprises the following steps:

[0053] (S. 1) Adjust the pH of whey protein with a concentration of 4.0-6.0wt% to 3.5-4 with 1M citric acid, heat in a water bath at 30-40℃ for 60-80min, centrifuge to obtain the supernatant, add 7.0-7.5wt% sodium chloride for salting out for 50-70min, centrifuge to collect the supernatant, repeat once, dialyze the solution for 48h, and freeze-dry for later use;

[0054] (S. 2) dissolving the β-lactoglobulin powder separated in step (S. 1) in deionized water to obtain a 1 wt% β-lactoglobulin solution, subjecting the solution to high-pressure microfluidization treatment at a pressure of 100 MPa, and performing a cycle operation for 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin;

[0055] (S. 3) Add 4% (w / w) succinic anhydride to the β-lactoglobulin solution modified by high-pressure microfluidization in step (S. 2), and stir in a water bath under constant temperature magnetic stirring. The pH of the acylation reaction is always maintained at 8-8.5, and the stirring is performed for 120-150 minutes to form succinylated β-lactoglobulin. The excess succinic anhydride and impurities are removed by dialysis for 48 hours to obtain a purified succinylated β-lactoglobulin solution. The solution is subjected to vacuum freeze-drying to obtain a powdered sample, and after grinding, uniform composite modified β-lactoglobulin particles are obtained.

[0056] Example 3

[0057] A method for preparing a composite modified β-lactoglobulin comprises the following steps:

[0058] (S. 1) Adjust the pH of whey protein with a concentration of 4.0-6.0wt% to 3.5-4 with 1M citric acid, heat in a water bath at 30-40℃ for 60-80min, centrifuge to obtain the supernatant, add 7.0-7.5wt% sodium chloride for salting out for 50-70min, centrifuge to collect the supernatant, repeat once, dialyze the solution for 48h, and freeze-dry for later use;

[0059] (S. 2) dissolving the β-lactoglobulin powder separated in step (S. 1) in deionized water to obtain a 1 wt% β-lactoglobulin solution, subjecting the solution to high-pressure microfluidization treatment at a pressure of 50 MPa, and performing a cycle operation for 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin;

[0060] (S. 3) Add 10% (w / w) succinic anhydride to the β-lactoglobulin solution modified by high-pressure microfluidization in step (S. 2), and stir in a water bath under constant temperature magnetic stirring. The pH of the acylation reaction is always maintained at 8-8.5, and the stirring is performed for 120-150 minutes to form succinylated β-lactoglobulin. The excess succinic anhydride and impurities are removed by dialysis for 48 hours to obtain a purified succinylated β-lactoglobulin solution. The solution is subjected to vacuum freeze-drying to obtain a powdered sample, and after grinding, uniform composite modified β-lactoglobulin particles are obtained.

[0061] Example 4

[0062] A method for preparing a composite modified β-lactoglobulin comprises the following steps:

[0063] (S. 1) Adjust the pH of whey protein with a concentration of 4.0-6.0wt% to 3.5-4 with 1M citric acid, heat in a water bath at 30-40℃ for 60-80min, centrifuge to obtain the supernatant, add 7.0-7.5wt% sodium chloride for salting out for 50-70min, centrifuge to collect the supernatant, repeat once, dialyze the solution for 48h, and freeze-dry for later use;

[0064] (S. 2) dissolving the β-lactoglobulin powder separated in step (S. 1) in deionized water to obtain a 1 wt% β-lactoglobulin solution, subjecting the solution to high-pressure microfluidization treatment at a pressure of 100 MPa, and performing a cycle operation for 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin;

[0065] (S. 3) adding 10% (w / w) succinic anhydride to the β-lactoglobulin solution modified by high-pressure microfluidization in step (S. 2) and subjecting the mixture to constant temperature magnetic stirring in a water bath, maintaining the pH of the acylation reaction at 8-8.5, stirring for 120-150 minutes, forming succinylated β-lactoglobulin, and performing dialysis treatment for 48 hours to remove excess succinic anhydride and impurities to obtain a purified succinylated β-lactoglobulin solution, subjecting the solution to vacuum freeze-drying to obtain a powdered sample, and grinding to obtain uniformly-composite modified β-lactoglobulin particles.

[0066] Example 5

[0067] A method for preparing a composite modified β-lactoglobulin comprises the following steps:

[0068] (S. 1) Adjust the pH of whey protein with a concentration of 4.0-6.0wt% to 3.5-4 with 1M citric acid, heat in a water bath at 30-40℃ for 60-80min, centrifuge to obtain the supernatant, add 7.0-7.5wt% sodium chloride for salting out for 50-70min, centrifuge to collect the supernatant, repeat once, dialyze the solution for 48h, and freeze-dry for later use;

[0069] (S. 2) dissolving the β-lactoglobulin powder separated in step (S. 1) in deionized water to obtain a 1 wt% β-lactoglobulin solution, subjecting the solution to high-pressure microfluidization treatment at a pressure of 50 MPa, and performing a cycle operation for 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin;

[0070] (S. 3) Add 20% (w / w) succinic anhydride to the β-lactoglobulin solution modified by high-pressure microfluidization in step (S. 2), and stir in a water bath under constant temperature magnetic stirring. The pH of the acylation reaction is always maintained at 8-8.5, and the stirring is performed for 120-150 minutes to form succinylated β-lactoglobulin. The excess succinic anhydride and impurities are removed by dialysis for 48 hours to obtain a purified succinylated β-lactoglobulin solution. The solution is subjected to vacuum freeze-drying to obtain a powdered sample, and after grinding, uniform composite modified β-lactoglobulin particles are obtained.

[0071] Example 6

[0072] A method for preparing a composite modified β-lactoglobulin comprises the following steps:

[0073] (S. 1) Adjust the pH of whey protein with a concentration of 4.0-6.0wt% to 3.5-4 with 1M citric acid, heat in a water bath at 30-40℃ for 60-80min, centrifuge to obtain the supernatant, add 7.0-7.5wt% sodium chloride for salting out for 50-70min, centrifuge to collect the supernatant, repeat once, dialyze the solution for 48h, and freeze-dry for later use;

[0074] (S. 2) dissolving the β-lactoglobulin powder separated in step (S. 1) in deionized water to obtain a 1 wt% β-lactoglobulin solution, subjecting the solution to high-pressure microfluidization treatment at a pressure of 100 MPa, and performing a cycle operation for 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin;

[0075] (S. 3) Add 20% (w / w) succinic anhydride to the β-lactoglobulin solution modified by high-pressure microfluidization in step (S. 2), and stir in a water bath under constant temperature magnetic stirring. The pH of the acylation reaction is always maintained at 8-8.5, and the stirring is performed for 120-150 minutes to form succinylated β-lactoglobulin. The excess succinic anhydride and impurities are removed by dialysis for 48 hours to obtain a purified succinylated β-lactoglobulin solution. The solution is subjected to vacuum freeze-drying to obtain a powdered sample, and after grinding, uniform composite modified β-lactoglobulin particles are obtained.

[0076] Comparative Example 1

[0077] A method for preparing acylated β-lactoglobulin comprises the following steps:

[0078] Add 4% (w / w) succinic anhydride to a 1wt% β-lactoglobulin solution, stir magnetically for 60-80 minutes at 37°C to fully mix, use 1M NaOH to keep the reaction pH at 8-8.5 during stirring, continue magnetic stirring for 30-60 minutes after the pH stabilizes, and fully react to form an acylated β-lactoglobulin solution. The solution is placed in a dialysis bag made of a regenerated cellulose membrane with a molecular weight cutoff of 8-14 kDa, dialyzed at 4°C for 48 hours, and the water is changed every 6-7 hours. The dialyzed acylated β-lactoglobulin is then vacuum freeze-dried to obtain a powdered granular sample, which is ground to obtain a uniform powder for later use.

[0079] Comparative Example 2

[0080] A method for preparing acylated β-lactoglobulin comprises the following steps:

[0081] Add 10% (w / w) succinic anhydride to a 1wt% β-lactoglobulin solution, stir magnetically for 60-80 minutes at 37°C to fully mix, use 1M NaOH to keep the reaction pH at 8-8.5 during stirring, continue magnetic stirring for 30-60 minutes after the pH stabilizes, and fully react to form an acylated β-lactoglobulin solution. The solution is placed in a dialysis bag made of a regenerated cellulose membrane with a molecular weight cutoff of 8-14kDa, dialyzed at 4°C for 48h, and the water is changed every 6-7h. The dialyzed acylated β-lactoglobulin is then vacuum freeze-dried to obtain a powdered granular sample, which is ground to obtain a uniform powder for later use.

[0082] Comparative Example 3

[0083] A method for preparing acylated β-lactoglobulin comprises the following steps:

[0084] Add 20% (w / w) succinic anhydride to a 1wt% β-lactoglobulin solution, stir magnetically for 60-80 minutes at 37°C to fully mix, use 1M NaOH to keep the reaction pH at 8-8.5 during stirring, continue magnetic stirring for 30-60 minutes after the pH stabilizes, and fully react to form an acylated β-lactoglobulin solution. The solution is placed in a dialysis bag made of a regenerated cellulose membrane with a molecular weight cutoff of 8-14 kDa, dialyzed at 4°C for 48 hours, and the water is changed every 6-7 hours. The dialyzed acylated β-lactoglobulin is then vacuum freeze-dried to obtain a powdered granular sample, which is ground to obtain a uniform powder for later use.

[0085] Comparative Example 4

[0086] A method for preparing high-pressure microfluidic modified protein comprises the following steps:

[0087] β-lactoglobulin powder was dissolved in deionized water to obtain a 1 wt% β-lactoglobulin solution. The solution was treated by high-pressure microfluidization at a pressure of 50 MPa and the operation was repeated 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin. A powder sample was obtained after vacuum freeze-drying, which was ground into uniform particles for later use.

[0088] Comparative Example 5

[0089] A method for preparing high-pressure microfluidic modified protein comprises the following steps:

[0090] β-lactoglobulin powder was dissolved in deionized water to obtain a 1 wt% β-lactoglobulin solution. The solution was treated by high-pressure microfluidization at a pressure of 100 MPa and the operation was repeated 3 times to obtain high-pressure microfluidization-modified β-lactoglobulin. A powder sample was obtained after vacuum freeze-drying, which was ground into uniform particles for later use.

[0091] Comparative Example 6

[0092] A method for preparing purified β-lactoglobulin comprises the following steps:

[0093] The whey protein with a concentration of 4.0-6.0wt% was adjusted to pH 3.5-4 with 1M citric acid, heated in a water bath at 30-40°C for 60-80min, and then centrifuged to obtain the supernatant. 7.0-7.5wt% sodium chloride was added for salting out for 50-70min, and then the supernatant was collected by centrifugation. The process was repeated once, and the solution was dialyzed for 48h and then freeze-dried for later use.

[0094]

Performance testing and analysis

[0095] [Test 1]

[0096] The β-lactoglobulin powder was dissolved in deionized water and magnetically stirred for 2 hours to completely hydrate it, thereby obtaining a uniformly dispersed β-lactoglobulin solution with a concentration of 0.5 wt%.

[0097] [Test 2]

[0098] According to the preparation methods of Examples 1 to 6 and Comparative Examples 1 to 6, composite modified β-lactoglobulin complex powder was selected and dissolved in deionized water for testing. Stir magnetically until completely hydrated, take 2 mL of 1 mg / mL sample solution and add 2 mL of 1.5 wt% ninhydrin solution, mix thoroughly, and immediately cool in an ice bath after boiling water bath at 95°C for 15 min, add 6 mL of 50% ethanol to terminate the reaction, and detect its absorbance value A1 at 570 nm ultraviolet light, and Comparative Example 6 is used as a blank control A0. The acylation degree of β-lactoglobulin at different mass ratios is calculated by the following calculation formula:

[0099]

[0100] The acylation degree of β-lactoglobulin modified by succinylation assisted by different pressures is shown in Figure 2. Figure 2 shown.

[0101] Figure 2 βLg, S4-βLg, S10-βLg, and S20-βLg are β-lactoglobulin samples with 0%, 4%, 10%, and 20% succinic anhydride, respectively. 0 MPa, 50 MPa, and 100 MPa represent the treatment of each sample with different high-pressure microfluidization pressures, where 0% and 0 MPa represent the single modified control groups of acylation reaction and high-pressure microfluidization, respectively. Figure 2 From the data comparison, it can be seen that: under the same pressure, the higher the mass fraction of succinic anhydride, the higher the acylation degree of the protein, that is, the more succinyl groups are bound to the surface of β-lactoglobulin. When the mass fraction of succinic anhydride is the same, the acylation degree of the β-lactoglobulin surface increases with the increase of pressure. The main reason is that high pressure causes the β-lactoglobulin structure to unfold, exposing more active sites on the surface, reducing the steric hindrance of the protein and promoting the binding of succinic anhydride and β-lactoglobulin. Compared with the sample solutions that have not been treated with pressure (Comparative Examples 1-3), the acylation degree of the sample solutions treated with pressure is significantly increased, among which the β-lactoglobulin sample solution with a pressure of 100 MPa and a mass fraction of 20% has the highest acylation degree.

[0102] [Test 3]

[0103] According to the preparation methods of Examples 1 to 6 and Comparative Examples 1 to 6, composite modified β-lactoglobulin complex powder was selected and dissolved in deionized water for testing. The particle size and potential of the sample solution were measured using a Zetasizer Pro nanoparticle size potential analyzer; the surface hydrophobicity of the protein in the sample solution was measured using bromophenol blue, 1 mL of 5 mg / mL sample solution was added with 20 μL of BBP reagent (1.0 mg / mL), the mixture was mixed thoroughly and reacted for 10 minutes, diluted 10 times to stop the reaction, and then the absorbance value A1 was measured at 595 nm using an ELISA reader. BBP reagent without sample was used as control A0; the free thiol content of protein was determined by adding sample protein to 10mM sodium phosphate buffer to prepare a 1% (w / v) sample solution, taking 1.5mL of sample solution and adding 7.5mL Tris-glycine buffer (containing 8M urea) and 40μL Ellman, mixing thoroughly and reacting at 25℃ in the dark for 1h, and finally measuring its absorbance value A2 at 412nm by microplate reader, D is the dilution factor, and C is the sample concentration. The surface hydrophobicity and free thiol content are calculated as follows:

[0104]

[0105]

[0106] The structural changes of β-lactoglobulin assisted by succinylation at different pressures are shown in Table 1:

[0107] In Table 1, βLg, S4-βLg, S10-βLg, and S20-βLg are β-lactoglobulin samples with succinic anhydride of 0%, 4%, 10%, and 20% mass fractions, respectively. 0 MPa, 50 MPa, and 100 MPa represent the treatment of each sample with different high-pressure microfluidization pressures, where the mass fractions of 0% and 0 MPa are the single modified control groups of acylation reaction and high-pressure microfluidization, respectively. From the data comparison in Table 1, it can be seen that: with the increase of pressure, the particle size of β-lactoglobulin decreases and the absolute potential decreases. The lower the particle size, the higher the solubility. Therefore, the solubility of protein increases after high-pressure treatment, but the decrease in absolute potential is not conducive to protein dissolution. After the high-pressure protein is acylated, its particle size increases significantly, due to the covalent bonding of succinyl and amino groups. However, compared with the group without high-pressure treatment, its particle size is still significantly reduced and the absolute potential increases. Therefore, the composite-treated β-lactoglobulin can improve its solubility more than the single modification treatment. As the pressure increases and the acylation reaction proceeds, the acyl group increases the net negative charge of the protein, resulting in a decrease in the non-polar groups on the protein surface and a decrease in the hydrophobicity of the protein surface. At the same time, a hydrophilic group is introduced, which changes the hydrophilic-hydrophobic ratio of the surface and improves the emulsifying activity of β-lactoglobulin. During the acylation process, the acylating agent can react with the free sulfhydryl group in addition to the ε-amino group of the protein to form a thioester bond or other covalent bond to stabilize the β-lactoglobulin protein molecule, thereby reducing the free sulfhydryl content during the modification process.

[0108] [Test 4]

[0109] According to the preparation methods of Examples 1 to 6 and Comparative Examples 1 to 6, 1 g of the composite modified β-lactoglobulin complex powder was selected and dissolved in 10 mL of Tris-HCl buffer solution. At room temperature, the mixture was stirred at 12000 rpm for 1 min and then centrifuged. The mixture was centrifuged at 10000 rpm for 30 min at 4°C, and the supernatant was collected. The β-lactoglobulin in the supernatant was determined by the biuret method. The solubility of β-lactoglobulin was expressed by the ratio of the β-lactoglobulin content in the supernatant after centrifugation to the β-lactoglobulin content in the solution before centrifugation. The experiment was repeated three times.

[0110]

[0111] The solubility of succinylated β-lactoglobulin assisted by different pressures is shown in Figure 2. Figure 3 shown.

[0112] Figure 3βLg, S4-βLg, S10-βLg, and S20-βLg are β-lactoglobulin samples with 0%, 4%, 10%, and 20% succinic anhydride, respectively. 0 MPa, 50 MPa, and 100 MPa represent the treatment of each sample with different high-pressure microfluidization pressures, where 0% and 0 MPa represent the single modified control groups for acylation reaction and high-pressure microfluidization, respectively. Figure 3 From the data comparison, it can be seen that: compared with the untreated sample group (Comparative Example 6), the solubility of the two groups of single modification treatments (Comparative Examples 1~5) has increased significantly, but the solubility of β-lactoglobulin after high-pressure microfluidization and acylation reaction has increased significantly. The structure of β-lactoglobulin after high-pressure microfluidization treatment is loose, the internal structure is open, and the particle size is reduced, which promotes its solubility increase; the acylation reaction causes the surface charge of β-lactoglobulin to change due to the combination of acyl groups, and the addition of acyl groups increases the intermolecular force, which promotes the increase in solubility. The combination of the two modifications significantly improves the modification effect. The high-pressure treatment unfolds the structure of β-lactoglobulin, increases the active sites, and promotes the protein acylation reaction. The increase in the degree of acylation significantly changes the surface charge and promotes hydration, thereby further increasing the solubility of β-lactoglobulin.

[0113] [Test 5]

[0114] The composite modified β-lactoglobulin complex particles were selected for testing according to the preparation methods of Examples 1 to 6 and Comparative Examples 1 to 6, respectively. 15 mL of 5 mg / mL sample solution was mixed with 5 mL of soybean oil, and homogenized at 10000 rpm for 4 min. 100 μL of sample solution was immediately drawn from the bottom of the homogenized emulsion and added to 9.9 mL of 0.1% (w / v) SDS solution. After fully mixing, the absorbance value A0 was measured under a 500 nm ultraviolet spectrophotometer. After standing for 30 min, 100 μL was drawn from the bottom again and added to 9.9 mL of SDS solution. After fully mixing, the absorbance value A30 was measured under a 500 nm ultraviolet spectrophotometer. Wherein, T is equal to 2.303, N is the dilution multiple, C is the protein sample concentration, θ is the oil phase volume fraction, and the emulsification activity of the sample solution is calculated by the following formula:

[0115]

[0116]

[0117] Emulsifying activity and emulsifying stability of β-lactoglobulin assisted by succinylation at different pressures Figure 4 shown.

[0118] Figure 4βLg, S4-βLg, S10-βLg, and S20-βLg are β-lactoglobulin samples with 0%, 4%, 10%, and 20% succinic anhydride, respectively. 0 MPa, 50 MPa, and 100 MPa represent the treatment of each sample with different high-pressure microfluidization pressures, where 0% and 0 MPa represent the single modified control groups for acylation reaction and high-pressure microfluidization, respectively. Figure 4 Comparison of the data shows that compared with the untreated sample group (Comparative Example 6) and the single modified group (Comparative Examples 1 to 5), the emulsification activity and emulsification stability of β-lactoglobulin treated with composite modification are significantly increased. The reason is that high pressure destroys the structure of β-lactoglobulin, exposes the internal hydrophobic groups, changes the surface hydrophobicity, and promotes the binding ability between protein and oil and water. At the same time, the internal active sites of the structure are exposed on the surface, which promotes the binding of acyl groups to it, thereby changing its surface hydrophilic-hydrophobic ratio and significantly increasing its emulsification activity. At the same time, the exposed hydrophobic groups and net charge promote the adsorption and stabilization of the protein at the oil-water interface, and promote the emulsification stability of β-lactoglobulin.

[0119] [Test 6]

[0120] The composite modified β-lactoglobulin complex particles were selected for testing according to the preparation methods of Examples 1 to 6 and Comparative Examples 1 to 6, respectively. 100 μL of 5 mg / mL sample solution was added to 100 μL of DPPH solution, mixed thoroughly, incubated in the dark at room temperature for 30 min, and then the absorbance value A1 was measured at 517 nm using an ELISA reader. The DPPH solution was used as a blank control A0. Each sample was measured three times in parallel, and the test was repeated three times.

[0121] The ABTS stock solution was diluted to an ABTS solution with an A0 of 0.7±0.02. 100 μL of 5 mg / mL sample solution was added to 200 μL of ABTS solution and vortexed for 30 seconds to mix thoroughly. After incubation in the dark at room temperature for 10 minutes, the absorbance value A1 was measured at 734 nm using a microplate reader. The ABTS solution was used as the blank control A0. Each sample was measured three times in parallel and the experiment was repeated three times.

[0122] Take 1mL of 5mg / mL sample solution and 3mL of 10mM pH6.6 sodium phosphate buffer, mix thoroughly, add 3mL of 1% (w / v) potassium ferrocyanide, react at 50℃ for 20min, and add 3mL (10%, w / v) trichloroacetic acid to the solvent after cooling to terminate the reaction. Centrifuge the solution at 4000rpm for 20min, collect the supernatant, take 2.5mL of the supernatant, add the same volume of deionized water and 0.5mL (0.1%, w / v) ferric chloride, mix thoroughly, incubate at room temperature for 10min, and measure its absorbance at 700nm with an enzyme marker.

[0123]

[0124]

[0125] The antioxidant capacity (DPPH free radical scavenging rate, ABTS free radical scavenging rate, reducing power) of β-lactoglobulin assisted by different pressures Figure 5 shown.

[0126] Figure 5 βLg, S4-βLg, S10-βLg, and S20-βLg are β-lactoglobulin samples with 0%, 4%, 10%, and 20% succinic anhydride, respectively. 0 MPa, 50 MPa, and 100 MPa represent the treatment of each sample with different high-pressure microfluidization pressures, where 0% and 0 MPa represent the single modified control groups for acylation reaction and high-pressure microfluidization, respectively. Figure 5 The data comparison shows that compared with the untreated sample group (Comparative Example 6) and the single modified group (Comparative Examples 1-5), the antioxidant activity of the β-lactoglobulin treated with the composite modification is significantly improved. The reason is that the high pressure exposes the internal structure of the protein on the surface, promoting the covalent bonding of the protein amino group and the acyl group. At the same time, the addition of the acyl group increases the reactivity of the protein and enhances the interaction with the antioxidant molecules, thereby promoting the antioxidant capacity of β-lactoglobulin.

[0127] [Test 7]

[0128] The composite modified β-lactoglobulin complex particles were tested according to the preparation methods of Examples 1 to 6 and Comparative Examples 1 to 6. 20 mL of 2% protein solution was taken, and its volume V0 was recorded. High-speed shearing was performed at 10000 rpm for 2 min, and the operation was repeated twice. The volume V1 was recorded, and the foaming activity of its β-lactoglobulin was calculated according to the following formula.

[0129]

[0130] The foaming activity of succinylated β-lactoglobulin assisted by different pressures Figure 6 shown.

[0131] Figure 6 βLg, S4-βLg, S10-βLg, and S20-βLg are β-lactoglobulin samples with 0%, 4%, 10%, and 20% succinic anhydride, respectively. 0 MPa, 50 MPa, and 100 MPa represent the treatment of each sample with different high-pressure microfluidization pressures, where 0% and 0 MPa represent the single modified control groups for acylation reaction and high-pressure microfluidization, respectively. Figure 6The data comparison shows that under the pressure treatment of high-pressure microfluidization, the foaming performance of β-lactoglobulin is significantly reduced. The reason is that the pressure changes the structure of the protein, reduces its surface tension, and thus reduces its foaming performance. At the same time, with the increase of acylation degree, its foaming activity has recovered. The reason is that acylation changes the charge of β-lactoglobulin, thereby weakening the interaction between molecules and being more conducive to the formation of a stable interface film.

[0132] The above embodiments are only used to illustrate the design ideas and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, any equivalent changes or modifications made based on the principles and design ideas disclosed by the present invention are within the protection scope of the present invention.

Claims

1. A method for preparing a composite modified β-lactoglobulin, characterized in that: The following steps are involved: (1) dissolving β-lactoglobulin powder in deionized water to form a β-lactoglobulin solution, and then subjecting the solution to microfluidization treatment to obtain a microfluidization-modified β-lactoglobulin solution; (2) The microfluidization-modified β-lactoglobulin solution is subjected to succinylation treatment to obtain microfluidization-assisted acylated β-lactoglobulin, and the acylated β-lactoglobulin solution is then dialyzed for purification, and finally freeze-dried to obtain composite modified acylated β-lactoglobulin.

2. The method for preparing a composite modified β-lactoglobulin according to claim 1, characterized in that: The preparation method of the β-lactoglobulin powder comprises: β-lactoglobulin is extracted from whey protein, dialyzed at room temperature, and then freeze-dried to obtain highly purified β-lactoglobulin powder.

3. The method for preparing a composite modified β-lactoglobulin according to claim 2, characterized in that: β-lactoglobulin is extracted from whey protein, dialyzed at room temperature, and then freeze-dried to obtain highly purified β-lactoglobulin powder, including: The pH of a whey protein solution with a concentration of 4.0-6.0wt% is adjusted to 3.5-4, heated in a water bath at 30-40°C for 60-80min, and then centrifuged to obtain the supernatant; 7.0-7.5wt% sodium chloride is added for salting out for 50-70min, and then the supernatant is collected by centrifugation, and the process is repeated once; the solution is dialyzed for 48h and then freeze-dried for use.

4. The method for preparing a composite modified β-lactoglobulin according to claim 3, characterized in that: The pH of the whey protein solution was adjusted to 3.5-4 with 1M citric acid.

5. The method for preparing a composite modified β-lactoglobulin according to claim 1, characterized in that: The concentration of the β-lactoglobulin solution in step (1) is 0.5-1.5 wt %.

6. The method for preparing a composite modified β-lactoglobulin according to claim 1, characterized in that: The pressure of the microfluidization treatment in step (1) is 50-100 MPa.

7. The method for preparing a composite modified β-lactoglobulin according to claim 1, characterized in that: The step (2) comprises: The β-lactoglobulin solution modified by microfluidization in step (1) is added with succinic anhydride at a concentration of 4% to 20% (w / w), and the solution is subjected to constant temperature magnetic stirring in a water bath. The pH of the acylation reaction is always maintained at 8 to 8.5, the temperature is 35 to 50° C., and the solution is stirred for 120 to 150 minutes to form a microfluidization-assisted acylated β-lactoglobulin solution. The solution is then dialyzed for 48 hours to remove excess succinic anhydride and impurities to obtain a purified microfluidization-assisted acylated β-lactoglobulin solution. The purified microfluidization-assisted acylated β-lactoglobulin solution is subjected to vacuum freeze-drying to obtain a powdered sample, i.e., a composite modified β-lactoglobulin.

8. The method for preparing a composite modified β-lactoglobulin according to claim 7, characterized in that: The pH was adjusted to 8-8.5 with 1-2 M sodium hydroxide solution.

9. The composite modified β-lactoglobulin prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the composite modified β-lactoglobulin prepared by the preparation method according to any one of claims 1 to 8 or the composite modified β-lactoglobulin according to claim 9 in protein processing function.

Images